ABSTRACT Next-generation sequencing (NGS) has the potential to revolutionize how infectious disease is diagnosed and treated, enabling the detection of a wide variety of pathogens in a single test. However, despite the increasing adoption of molecular diagnostics in clinical microbiology labs, NGS has not become widespread due to its cost, turnaround time, and the technical and computational expertise required to produce and analyze the data. To overcome these challenges, Arc Bio is developing a suite of technologies that will enable clinical microbiology labs to implement NGS pipelines. The Arc Bio Infectious Disease Pipeline (IDP) product includes all the experimental protocols and software necessary to go from sample to answer for pathogen detection in clinical samples, including novel tools to make pathogen sequencing less expensive, more sensitive, and more accurate. One of these tools is a Cas9-based host DNA/RNA depletion technology that was developed in Phase I of this project. Depletion of host sequences dramatically reduces the cost and increases sensitivity when sequencing primary samples such as plasma and cerebrospinal fluid. We have achieved the goals set forth in that proposal, including 99% depletion of rRNA and 50% depletion of abundant transcriptome sequences from RNAseq libraries; furthermore, we demonstrate 100-fold enrichment of HIV sequences in depleted samples, with no detectable off-target depletion. In Phase II, we will develop and validate our IDP toward sequencing samples from HIV-infected and immunocompromised individuals. This will involve the following activities: 1) optimizing and scaling up our host depletion technology; 2) completing development of our proprietary graph-based analytics software, which enables accurate strain-level detection that ?learns? as more data is acquired; and 3) validating our complete pipeline on samples from healthy individuals for baselining, as well as those from patients infected with HIV, HCV, and HBV. By performing unbiased next-generation sequencing with host depletion on these samples, we will be able to report not only viral genotype and drug resistance mutations, but also viral titer and co-infections (such as hepatitis B and C). The result of this project will be a complete, validated pipeline that will include all protocols and software necessary for initial deployment into alpha partner clinical labs.